Mini alkaline plasmid preparation with rayon extraction and spermine precipitation. (modified 2/18/06)

DNA is prepared from bacteria scraped off the surface of a petrie dish.  This preparation produces several micrograms of good quality DNA from the bacteria grown on 1/6 of a plate.  The procedure can easily be scaled-up for cells from half or a full plate.  The cells that remain on the plate after scraping can be used to inoculate cultures for large scale plasmid preparations, as long as there has not been any cross contamination.  The plates of cells can be stored in the refrigerator for several weeks.

*Note about DNA sequencing - it turns out that the spermine precipitation procedure described at the end of the protocol does not yield DNA that can be reliably sequenced by our facility.  An alternative procedure employing PEG precipitation has proven more reliable.

The following solutions must be set up before you start to process cells from 1/6 of a plate:

Procedure for isolating DNA that can be restriction cut.
  1. Mark each plate to divide it into 6 or 8 sectors.  The agar should contain the appropriate antibiotic.  Streak cells from a single colony throughout each sector.  Make a few streaks at the perimeter of the dish and then spread the cells from the edge to the center of the plate.  Grow cells overnight at 37oC.
  2. The next day, the cells can be processed immediately, or the plates can be stored for a few days in the refrigerator.
  3. Using a pair of clean tweezers that have been sterilized with ethanol, bend the end of a sterile yellow pipette tip so that it resembles a miniature hockey stick.  Use a separate tip for each sector of cells.  Carefully scrape up bacteria from a sector and place the yellow tip and cells in 100 ul of solution 1 in a 1.5 ml eppendorf tube.  Proceed onto other sectors.  The cells can sit in solution 1 for 30' to 60'.
  4. Agitate the yellow tips to disperse the cells.  Discard the yellow tips.
  5. Add 200 ul of solution 2 to each tube.  Cap each tube and mix by gently inverting the tube several times. Examine the tube when you invert it to be certain that the contents at the very bottom of the tube are being mixed.  Place tubes on ice for 5'.
  6. Add 150 ul of cold solution 3, mix by gently inverting several times and place on ice for 5'.
  7. Spin in microfuge for 5'.
  8. While the samples are spinning, assemble small rayon columns in yellow tips.  At a slight angle, clip approximately 2 mm off the end of one yellow tip per sample.  The slight angle keeps the tip open when it is resting against the bottom of a 1.7 ml microfuge tube.  Using another yellow tip, loosely stuff a wad of rayon (not cotton) into a yellow tip.  The plug should be approximately 0.5 cm in length when pushed close to the end of the yellow tip.  Place one rayon packed tip in each 1.7 ml microfuge tube.
  9. Add the supernatant from the solution III precipitation to the yellow tip and allow the supernatant to flow through the rayon by gravity.  It may be necessary to tap the yellow tip against the side of the 1.7 ml tube to initiate the flow. Also, you may have to lift up each yellow tip to get most of the sample to flow through the rayon.  The solution stops flowing when it reaches the rayon.  Discard the yellow tip with the residual solution.
  10. Add 1 ug of RNase A to each sample and digest at room temperature for 30'.
  11. Add 0.6 volumes of isopropanol, mix, and immediately spin the samples at 4oC for 10'.
  12. Discard the supernatant and rinse the pellet with cold 75% ethanol.
  13. Spin the samples for 5' and discard the supernatant.
  14. Dry the pellets and then dissolve them in 50 ul of TE.
  15. Subject DNA to restriction analysis.

Further purification of DNA for sequencing, subcloning, or fly transformation.
  1. Add 150 ul of TE + 0.1 M NaCl.
  2. Extract with 200 ul of leder phenol (phenol/chloroform/isoamyl alcohol).  Repeat the extractions one or two times more if there is a cloudy interface.  Ether extract to remove residual phenol.  Float the tubes in warm water with the caps off so the ether evaporates.
  3. Add 10 ul of 100 mM spermine, mix, and chill on ice for 15 minutes.
  4. Spin 15' at 4oC.
  5. Discard supernatant and dissolve the DNA in 25 ul of 0.6 M Na Acetate (pH 6.0) {high salt is required to dissociate the spermine from the DNA}.   Agitate the sample for at least 10' to be certain the DNA dissolves.
  6. Add 25 ul H2O and 125 ul of cold, 100% ethanol, mix, and ice for 15'.
  7. Spin 15' at 4oC.
  8. Discard supernatant and rinse pellet with 100 ul of cold, 75% ethanol.  Spin 5' and discard supernatant.
  9. Dissolve DNA in 20 ul of 1/10X TE  (the reduced level of EDTA is so the DNA can be sequenced).
  10. Determine the DNA concentration with the fluorometer.